Evidence for neurotoxicity and oxidative stress in zebrafish embryos/larvae treated with HFPO-DA ammonium salt (GenX).

"GenX" [ammonium perfluoro (2-methyl-3-oxahexanoate] was developed as a replacement chemical for toxic perfluorinated compounds to be used in product manufacturing. Here, we assessed developmental, mitochondrial, and behavioral toxicity endpoints in zebrafish embryos/larvae exposed to GenX. GenX exerted low toxicity to zebrafish embryos/larvae up to 20 mg/L. GenX did not affect mitochondrial oxidative phosphorylation nor ATP levels. ROS levels were reduced in larvae fish exposed to 10 and 100 µg/L, indicative of an antioxidant defense; however, ROS levels were elevated in fish exposed to 1000 µg/L. Increased expression of cox1 and sod2 in GenX exposed 7-day larvae was noted. GenX (0.1 or 1 µg/L) altered transcripts associated with neurotoxicity (elavl3, gfap, gap43, manf, and tubb). Locomotor activity of larvae was reduced by 100 µg/L GenX, but only in light periods. Perturbations of anxiety-related behaviors in larvae were not observed with GenX exposure. These data inform risk assessments for long-lived perfluorinated chemicals of concern.

The novel insecticide broflanilide dysregulates transcriptional networks associated with ion channels and induces hyperactivity in zebrafish (Danio rerio) larvae.

Broflanilide is a novel insecticide that is classified as a non-competitive γ-aminobutyric acid (GABA) receptor antagonist. However, indiscriminate use can have negative effects on non-target species. The objective of this study was to determine the sub-lethal toxicity potential of broflanilide in early staged zebrafish. Embryos/larvae were assessed for multiple molecular and morphological endpoints following exposure to a range of concentrations of broflanilide. The insecticide did not affect hatch rate, the frequency of deformities, nor did it impact survival of zebrafish at exposure concentrations up to 500 µg/L over a 7-day period from hatch. There was also no effect on oxidative consumption rates in embryos, nor induction of reactive oxygen species in fish exposed up to 100 µg/L broflanilide. As oxidative stress was not prominent as a mechanism, we turned to RNA-seq to identify potential toxicity pathways. Gene networks related to neurotransmitter release and ion channels were altered in zebrafish, consistent with its mechanism of action of modulating GABA receptors, which regulate chloride channels. Noteworthy was that genes related to the circadian clock were induced by 1 µg/L broflanilide exposure. The locomotor activity of larval fish at 7 days was increased (i.e., hyperactivity) by broflanilide exposure based on a visual motor response test, corroborating expression data indicating neurotoxicity and motor dysfunction. This study improves the current understanding of the biological responses in fish to broflanilide exposure and contributes to risk assessment strategies for this novel pesticide.

Dataset for diseases associated with exposure to broflanilide, a novel pesticide, in larval zebrafish (Danio rerio).

Broflanilide is a novel pesticide that can antagonize ion channels and disrupt neurotransmitter systems in the brain. Zebrafish larvae were exposed to either 0, 1 or 10- µg/L broflanilide in the water for a period of 7 days during early development. RNA extraction was conducted on larval zebrafish for RNA-seq analysis using the Illumina NovoSeq 6000. Raw sequence data were processed through fastp and clean reads obtained by removing adapter and poly-N sequences. Alignment and differential gene expression analysis was conducted using HISAT2, StringTie assembler, and FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced). Subnetwork enrichment analysis (SNEA) revealed that exposure to 1 µg/L broflanilide altered gene networks associated with axonal injury, depression, neuroinflammation, and traumatic brain injury while exposure to 10- µg/L broflanilide resulted in changes in gene networks associated with brain infarction and ischemia, excitotoxicity, and neurogenic inflammation. In addition, genes related to MPTP-induced neurotoxicity were altered by broflanilide which has relevance for Parkinson's disease. Several transcripts were identified as being associated with a disease network link to neurodegeneration and included phospholipase A2 activating protein, calpain 1, ATPase Na+/K+ transporting subunit alpha 2, glia maturation factor beta, sphingomyelin phosphodiesterase 1, leucine rich repeat kinase 2, glutamate ionotropic receptor NMDA type subunit 2C, lysosomal associated membrane protein, and calcium/calmodulin dependent protein kinase II alpha among others. Data presented here include disease biomarkers for a novel pesticide and can be reused to refine models that describe adverse outcome pathways for neurotoxicity.

Molecular and behavioral toxicity assessment of tiafenacil, a novel PPO-inhibiting herbicide, in zebrafish embryos/larvae.

Tiafenacil is a newly registered herbicide and a protoporphyrinogen IX oxidase inhibitor. However, sub-lethal effects of PPO-inhibitors in aquatic species are unknown. Embryos or larvae were exposed to 0.1 µg/L up to 10 mg/L tiafenacil for 7-days post-fertilization. Decreased survival (> 50%) and deformities were noted at concentrations > 1 mg/L. Potency (EC50) of tiafenacil for 5- and 7-day larvae were 818.1 µg/L and 821.7 µg/L, respectively. Pericardial and yolk sac edema were the most frequent deformities observed. Heartbeat frequency at 3 dpf was decreased in zebrafish exposed to > 10 µg/L tiafenacil, coinciding with increased reactive oxygen species. Oxygen consumption rates were not affected by tiafenacil, nor did we detect differences in indicators of apoptosis. The abundance of eighteen transcripts related to oxidative stress and mitochondrial complexes I through V were unchanged. Larval activity was decreased with exposure to 1000 µg/L tiafenacil. These data contribute to risk assessment for a new class of herbicide.